Early studies leading to the development of high-performance capillary electrophoresis (HPCE) were those of Jorgenson and Lukacs. Jorgenson, J. W., et al., Anal. Chem. 53:1298 (1981); Jorgenson, J. W., et al., Clin. Chem. 27:1551 (1981); Jorgenson, J. W., et al., J. Chromatogr. 218:209 (1981). The experiments reported in these studies demonstrated that extremely high efficiencies in the separation of ionic molecules can be achieved in a narrow-bore capillary, i.e., one of less than 100 microns inner diameter, by applying a high, constant voltage between the two ends of the capillary, thereby causing the molecules to migrate toward one of the electrodes in discrete zones according to molecular weight, and permitting detection of the zones by focusing a sufficiently sensitive detector on a point in the migration path. The basic experimental arrangement of Jorgenson, et al. has since been used in the separation of various types of molecules, both small and large, in a highly efficient manner. In some systems, the electrophoretic principle serves as the basis for these separations. In others, the separations are the result of electroosmotic action in micellar or other buffer-modified systems. See, Tsuda, T., et al, J. Chromatogr. 264:385 (1983); Terabe, S. et al., Anal. Chem. 56:111 (1984); Terabe, S., et al., Anal. Chem. 57:834 (1985); and Foley, J. P., Anal. Chem. 62:1302 (1990). Still other systems rely on the electroosmotic action on microemulsions. Watarai, H., Chem. Lett. (Japan) 391 (1991). Application of the method to highly charged polymers, such as oligo- and polynucleotides, was demonstrated by Cohen, A. S., et al., Proc. Natl. Acad. Sci. U.S.A. 85:9660 (1988), who report the separation of such polymers in chemically immobilized gels such as crosslinked polyacrylamides. Because of these and additional developments, HPCE is now a highly successful and widely commercialized instrumental analytical method.
The choice of separation medium to be used inside a capillary in HPCE is often fundamental to the success of a given separation problem. As a result, many investigators have discovered that by careful selection and modification of the separation medium one can achieve separations of components which are otherwise difficult to separate. This has led, for example, to the use of cyclodextrins, micellar systems and chiral gels for the separations of chiral entities. Fanali, S., J. Chromatogr. 474:441 (1989); Liu, J. et al., J. Chromatogr. 519:189 (1990); Terabe, S., et al. J. Chromatogr. 516:23 (1990); Guttman, A. et al., J. Chromatogr. 448:41 (1988). Other examples are the separations of proteins in surface-coated open tubes. Hjerten, S., J. Chromatogr. 347:1991 (1985); Cobb, K. A., et al., Anal. Chem. 62:2478 (1990); Wiktorowicz, J. E., et al, Electrophoresis 11:769 (1990); Town, J. K., et al., J. Chromatogr. 516:69 (1990). Still further examples are the separation of oligosaccharides in highly concentrated, immobilized gels. Liu, J., et al., J. Chromatogr. 559:223 (1991).
Nevertheless, large biopolymers, such as polysaccharides, certain proteins, or fragments of nucleic acids, do not respond well to attempts at capillary electrophoresis, either in open tubes filled with buffer solution or in tubes containing immobilized gels. In buffer-filled open tubes, large biopolymers tend to migrate together. In gel-filled capillaries, the pore structures of the gels are resistant to penetration by very large molecules, thereby interfering with the successful movement of the molecules past the detector. On the other hand, successful separations have been achieved in some cases by using separation media consisting of viscous solutions of polymers, such as water-soluble derivatives of cellulose, low-melting agarose, galactomannan, linear polyacrylamides, polyethylene glycols, and polyvinyl alcohols. Zhu, M. D., et al., U.S. Pat. No. 5,089,111 (Feb. 18, 1992); Grossman, P. D., et al., Biopolymers 31:1221 (1991). The polymeric solutes in these solutions form networks of aggregates which are sufficiently flexible to yield to the large polyionic species sought to be separated as these species migrate under the influence of an electric field.
A property of very large biopolymers such as chromosomal DNA is their tendency to stretch in an electric field, exhibiting what is known in the literature as "reptation." De Gennes, P. G., et al., Scaling Concepts in Polymer Physics, Cornell University Press, Ithaca, N.Y. (1979); Lerman, L. S., et al., Biopolymers 21:995 (1982); Lumpkin, O. J., et al., Biopolymers 24:1573 (1985). Reptation causes molecules of vastly different chain lengths to migrate through the separation medium network at very similar speeds, spoiling the attempt at separation. This phenomenon was recognized and turned to advantage by Schwartz, D. C., et al., Cell 37:67 (1984), who designed an experiment in which these large molecules were subjected to alternating fields at an angle relative to each other and to the direction of migration. During the alternating, or "pulsing," of the fields in this manner, the molecular distortions in the form of shape reorientations and the formation of kinks or "hernias" which occur during reptation alternate with molecular relaxation, and the rates at which the distortion and relaxation occur vary with the sizes of the molecules in question. These differing rates of response within each portion of the alternation cycle result in differing rates of the overall or averaged migration for each species, which in turn results in a separation of the species on the basis of their molecular weight.
In the various studies which have since been reported, the alternations in the electric field have been performed by switching between fields oriented at angles relative to each other, as well as between fields which are rotated a full 180.degree. relative to each other, the latter commonly referred to as "field inversion." Like the studies using angled fields, studies involving field inversion have largely been performed in slab-shaped media, and most such studies have involved the separation of molecules of the size of very large DNA chains, i.e., approximately 10.sup.5 to 10.sup.7 bases in size (10.sup.7 to 10.sup.9 daltons). Little or no effect has been observed in attempts to separate smaller molecules by these techniques. The separation medium most widely used has been low-melting agarose. Schwartz, D. C., et al., Cell 37:67 (1984); Carle, G. F., et al., Science 232:65 (1986). Fields strengths most commonly used are within the range of 2 to 10 V/cm, with separations taking hours, and even days.
An attempt at the use of field inversion (or "pulsed fields") in capillaries is reported by Heiger, D. N., et al., J. Chromatogr. 516:33 (1990). The major portion of the experimental results reported by these authors was obtained using continuous (nonalternating) fields rather than pulsed fields, however, and the only attempts at pulsed-field separations involved a mixture of only two double-stranded DNA fragments, each of which had a molecular weight well in excess of 2,000,000, and for which the authors had already exhibited complete resolution in a continuous-field experiment. In two of the pulsed-field experiments, the peak separation was no greater than that achieved in the continuous-field experiment. An increase in peak separation was achieved when the pulsing frequency was varied, but the maximum improvement over a total of eight attempts was only a 25% increase in the separation. Other than this, no successful application of the pulsed-field technique to HPCE has been reported. This may have been due to experimental difficulties involved in pulsing at the high voltages normally used in HPCE (typically 100 to 300 V/cm), the perceived importance of pulsing angles other than 180.degree. C., or difficulties in the selection of such parameters as appropriate column matrices and sample concentrations.
It is for these reasons that the present invention is surprising and unexpected in view of the state of the art.